Arenavirus reverse genetics: implications for anti-arenavirus drugs We have developed a novel and powerful reverse genetics system to investigate the cis-acting sequences and trans-acting factors that control prototypic arenavirus LCMV replication, gene expression, assembly and budding. We (23) and others (33, 38) have shown that Z is the driving force behind arenavirus budding in a process mediated by late (L) domain motifs at the C-terminus of Z (PTAP and PPPY), which direct virus budding via interaction with specific host cell proteins (10). Moreover, we succeeded in rescuing infectious LCMV entirely from cloned cDNAs (9, 29). The rescue of rLCMVwith predetermined genetic alterations has opened new avenues to investigate arenavirus-host interactions and associated disease, uncover specific steps of the virus life cycle amenable to targeting, and identify potential antiviral compounds. PSWRCE-supported studies already have generated new concepts for strategies against arenavirus, briefly: a) Targeting the arenavirus promoter via aminoglucoside-based drugs. We documented that the activity of the arenavirus genomic promoter recognized by the virus polymerase requires both: i) sequence specificity within the highly conserved 3'-terminal 19 nt of arenavirus genomes, and 2) the integrity of the predicted panhandle structure formed via sequence complementarity between the s'-and s'-termini of the viral genome RNA This RNA element presents a new potential target for anti-arenavirus drugs based on the use of aminoglycoside-based compounds (34, 35,42). Subsequent to obtaining evidence that several arginine conjugate aminoglycosides AAC exhibit a powerful inhibitory effect on LCMV multiplication, we are currently examining the antiviral value of aminoglycosides in a mouse model of arenavirus infection. b) Targeting arenavirus budding. Z-mediated budding of arenaviruses requires its interaction with specific cellular proteins including members of the endosomal/MVB pathway. We have identified TSGioi and CHMP3, respectively members of the ESCRT-I and ESCRT-III complexes, as Z-interacting cellular proteins required for arenavirus budding.